The major goals of this project are to understand basic aspects of virulence and host immunity to lipopolysaccharide (LPS)-smooth strains of P. aeruginosa that are major causes of hospital acquired pneumonia and community-acquired bacterial keratitis of the eye. The primary hypothesis to be evaluated is that effective immunity to P. aeruginosa will require the use of immunogens that elicit both antibodies and cytotoxic T cells (CTL) that can kill extracellular and intracellular P. aeruginosa. In regard to immunity to the extracellular stage of pathogenesis, for many P. aeruginosa strains, effective humoral immunity requires antibodies that recognize highly variable subtype-specific epitopes on the LPS O side chain. Subtype epitope variability greatly increases the number of antigens that need to be incorporated into a vaccine that go beyond the 20 major LPS-based serogroups of P. aeruginosa. More problematic has been the difficulty in inducing such antibodies by LPS-O antigen based immunogens. Appropriate LPS-specific antibodies cannot readily be elicited with polyvalent vaccines because of antagonistic interactions among structurally-related immunogens and the host immune system, so newer antigen-delivery systems are needed. To address this need we are focusing on producing and testing live, attenuated P. aeruginosa vaccines that would be better immunogens for inducing LPS specific protective antibody and also provide immunity to the intracellular phase of P. aeruginosa infection that has recently been documented. P. aeruginosa enters epithelial cells via the cystic fibrosis transmembrane conductance regulator (CFTR) during the course of lung and eye infections, and epithelial cell ingestion has a significant impact on the organism's ability to cause infections. The major aim of the proposal will be to explore how attenuated vaccines that enter lung epithelial cells prior to clearance induce protective antibodies and T cells that recognize infected epithelial cells and kill the intracellular P. aeruginosa. Overall, the aims are designed to gain a better understanding of how to elicit protective immunity to P. aeruginosa, focusing on use of attenuated strains to provoke humoral and cell-mediated immunity (CMI) against both cytotoxic and noncytotoxic P. aeruginosa strains. Specific questions to be addressed include: 1) can attenuated strains of P. aeruginosa be safely produced that elicit humoral and cellular immune effectors that kill P. aeruginosa outside and inside of epithelial cells?; 2) can such vaccines elicit broadly-based acquired resistance to P. aeruginosa infection; and, 3) how is CMI elicited by attenuated vaccines and how does this immune effector contribute to resistance to infection? The results of this work should further our insights into P. aeruginosa pathogenesis, development of acquired immunity and the potential to develop effective reagents for active and passive immunization to augment the prevention and treatment off, aeruginosa infections.